Culture system and culture method for thermophilic bacteria biofilm in flowing dairy product matrix

Abstract

The invention relates to a culture system and a culture method for a thermophilic bacteria biofilm in a flowing dairy product matrix, and belongs to the technical field of food. The culture system comprises a milk supply tank used for storage and supply of dairy products; the power device is connected with the milk supply tank and is used for conveying the dairy products in the milk supply tank to the biological membrane culture unit; the biological membrane culture unit comprises a tubular reactor and an annular inner wall stainless steel layer arranged in the tubular reactor; a sample placing frame for placing a sample wafer is arranged on the inner surface of the stainless steel layer around the inner wall; the input end of the biofilm culture unit is connected with the power device, and the output end is connected with the one-way valve; the one-way valve is used for controlling the single flow direction of dairy products; the sampling port is connected with the one-way valve and used for controlling dairy products to flow to the sampling bottle or the waste liquid recovery tank. The thermophilic bacteria biofilm cultured by the system not only can be used for counting the total number of bacterial colonies, but also can be used for observing a laser confocal microscope marked by a specific fluorescent probe.

Description

Technical Field

[0001] The present invention belongs to the technical field of food, and particularly relates to a culture system and a culture method for thermophilic bacteria biofilm in a flowing dairy matrix. Background Art

[0002] Thermophilic bacteria are common but troublesome spoilage bacteria in the food processing industry, especially in dairy processing, and have a particularly significant impact on pasteurized milk and milk powder products. Pasteurization cannot completely kill thermophilic bacteria and their spores, and some spores can even tolerate ultra-high temperature short-time sterilization. Milk contains rich nutrients such as lactose that can promote the formation of bacterial biofilms. Thermophilic bacteria with the ability to form biofilms can adhere to the inner walls of equipment such as pipelines, heat exchangers, and evaporators, and it is difficult to completely remove them by conventional cleaning methods. In the evaporation and concentration unit during the processing of milk powder, a high-temperature environment of 50°C - 70°C is usually adopted, but this suitable growth temperature condition further promotes the rapid reproduction and colonization of thermophilic bacteria, making the biofilm gradually become a cross-contamination source in the production line, thereby damaging product quality and leaving potential food safety hazards.

[0003] Although the harm of thermophilic bacteria biofilm has attracted the attention of the field of food microbiological detection, currently, the culture and detection of biofilm in the dairy system use static non-flowing skim milk as the nutrient matrix. However, due to the growth of thermophilic bacteria leading to the deterioration of milk, situations such as pH change, flocculation, stratification, nutrient consumption, and release of spoilage enzymes occur in the milk liquid, and these adverse conditions are continuously accumulated and amplified in the non-renewed culture medium. Therefore, static culture research cannot accurately reproduce the environment for the formation of thermophilic bacteria biofilm during dairy processing, and there is a huge difference between the design of such detection conditions and the requirements of actual production situations.

[0004] Therefore, the research on thermophilic bacteria biofilm in a flowing dairy matrix is an urgent problem to be solved at present and has important significance. Summary of the Invention

[0005] For this reason, the technical problem to be solved by the present invention is to overcome the problems of dairy product deterioration, culture medium spoilage, and product accumulation interfering with the formation of thermophilic bacteria biofilm in the conventional static biofilm culture method in the prior art.

[0006] To solve the above technical problems, the present invention provides a culture system and a culture method for thermophilic bacteria biofilm in a flowing dairy matrix.

[0007] The first object of the present invention is to provide a culture system for thermophilic bacteria biofilm in a flowing dairy matrix, including:

[0008] A milk supply tank: for storing and supplying dairy products; a power device, connected to the milk supply tank, for transporting the dairy products in the milk supply tank to the biofilm culture unit;

[0009] Biofilm culture unit: It includes a tubular reactor and a stainless steel layer on the inner wall of the ring provided in the tubular reactor; a sample placing rack for placing samples is provided on the inner surface of the stainless steel layer on the inner wall of the ring; the input end of the biofilm culture unit is connected to the power device, and the output end is connected to a one-way valve; both ends of the tubular reactor are provided with a first disassembly port and a second disassembly port for placing and sampling samples; the area of the stainless steel layer on the inner wall of the ring is equivalent to the inner surface area of the tubular reactor;

[0010] One-way valve: It is used to control the single flow direction of dairy products; a sampling port is connected to the one-way valve and is used to control the flow direction of dairy products to the sampling bottle or the waste liquid recovery tank.

[0011] In an embodiment of the present invention, it further includes a water bath heating device and a heating jacket heating device; the water bath heating device is used to heat the milk supply tank; the heating jacket heating device includes a flexible heating jacket and a temperature control device, and the flexible heating jacket is wrapped on the surface of the tubular reactor; the heating jacket heating device is used to heat the tubular reactor.

[0012] In an embodiment of the present invention, the inner diameter of the tubular reactor is 2 cm - 6 cm, and the length is 15 cm - 30 cm;

[0013] The shape of the sample is circular or square. The diameter of the circular sample is 1 cm - 2 cm, and the side length of the square sample is 1 cm - 2 cm.

[0014] In an embodiment of the present invention, the material of the sample is stainless steel, plastic or glass.

[0015] In an embodiment of the present invention, the sample placing rack is provided with independently detachable or row-connected non-detachable grooves, and the shape of the grooves matches the shape of the sample.

[0016] In an embodiment of the present invention, the biofilm culture unit is in an inclined state, and the inclination angle is 10° - 45°, which is achieved by a low support and a high support.

[0017] The second object of the present invention is to provide a method for culturing thermophilic bacteria biofilm in a flowing dairy product matrix, using the described culture system as a generating device, including the following steps:

[0018] S1. Place the first sample on the sample placing rack and install it in the biofilm culture unit;

[0019] S2. Load the dairy product containing thermophilic bacteria into the milk supply tank and set the heating temperatures of the water bath heating device and the heating jacket heating device;

[0020] S3. Transport the dairy product containing thermophilic bacteria to the biofilm culture unit through the power device for culturing, and take out the first sample;

[0021] S4. Re-prepare the culture system, place the second sample piece and the first sample piece on the sample rack, and install them into the biofilm culture unit;

[0022] S5. Load the dairy product without thermophilic bacteria into the milk supply tank, and set the heating temperatures of the water bath heating device and the heating jacket heating device;

[0023] S6. Transport the dairy product without thermophilic bacteria to the biofilm culture unit for cultivation through the power device, take out the first sample piece to measure the biofilm shedding situation; and / or, take out the second sample piece to measure the degree of influence of the second sample piece by the first sample piece.

[0024] In one embodiment of the present invention, in S2, the thermophilic bacteria are selected from one or more of Bacillus licheniformis, Anaerobacillus thermophilus, Geobacillus stearothermophilus, and Bacillus subtilis.

[0025] In one embodiment of the present invention, in S2, the dairy product containing thermophilic bacteria also needs to be subjected to strain activation treatment before use; the strain activation is carried out by shaking culture at 54°C - 56°C for 6h - 10h.

[0026] In one embodiment of the present invention, the heating temperatures of the water bath heating device and the heating jacket heating device are independently 50°C - 70°C.

[0027] In one embodiment of the present invention, the conveying speed of the power device is independently 1mL / min - 100mL / min.

[0028] In one embodiment of the present invention, the cultivation time is independently 6h - 24h.

[0029] The technical solution of the present invention has the following advantages compared with the prior art:

[0030] (1) The culture system described in the present invention can realize the formation process of thermophilic bacteria biofilm on the surface of sample pieces in a dairy product production line under the condition of dynamic update of the culture medium. It has the characteristics of good controllability and wide application scenarios, providing ideas for the research of bacterial biofilms in dynamic nutrient matrices.

[0031] (2) The thermophilic bacteria biofilm obtained by the culture method described in the present invention can not only be used for colony count, but also for observation by a laser confocal microscope with specific fluorescent probe labeling. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] In order to make the content of the present invention easier to be clearly understood, the following further describes the present invention in detail according to the specific embodiments of the present invention in combination with the drawings, wherein:

[0033] Figure 1Schematic structural diagram of the culture system for thermophilic bacteria biofilms in flowing dairy substrates of the present invention;

[0034] Figure 2 Schematic diagram of the sample placement rack in the culture system for thermophilic bacteria biofilms in flowing dairy substrates of the present invention; wherein, (A) are independent and separable circular grooves or square grooves, and (B) are row-connected and inseparable circular grooves or square grooves;

[0035] Figure 3 Cultivation process of the thermophilic bacteria biofilms of the present invention;

[0036] Figure 4 Application of the thermophilic bacteria biofilms of the present invention in the total colony count; wherein, (A) is the ability of thermophilic bacteria to form biofilms on the stainless steel surface, (B) is the shedding of mature thermophilic bacteria biofilms, and (C) is the secondary adhesion ability of thermophilic bacteria biofilms;

[0037] Figure 5 Application of the thermophilic bacteria biofilms of the present invention in the observation by a laser confocal microscope with specific fluorescence probe labeling;

[0038] Explanation of reference numerals: 1 - water bath heating device, 2 - milk supply tank, 3 - power device, 4 - flexible heating sleeve, 5 - tubular reactor, 6 - stainless steel sample piece, 7 - sample placement rack, 8 - stainless steel layer on the inner wall of the ring, 9 - low support, 10 - high support, 11 - first disassembly port, 12 - second disassembly port, 13 - one-way valve, 14 - sampling port, 15 - waste liquid recovery tank, 16 - hose, 17 - heat insulation layer, 18 - thermophilic bacteria biofilm. Detailed implementation manners

[0039] The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments, so that those skilled in the art can better understand the present invention and be able to implement it, but the examples given are not intended to limit the present invention.

[0040] In the present invention, unless otherwise specified, the technical and scientific terms used in the present invention have the same meanings as those generally understood by those skilled in the technical field to which the present invention belongs.

[0041] In the present invention, unless otherwise specified, the term "and / or" includes any and all combinations of one or more of the related listed items.

[0042] In the present invention, unless otherwise specified, the experimental methods used in the embodiments of the present invention are all conventional methods without special instructions, and the materials, reagents, etc. used can be obtained from commercial channels without special instructions.

[0043] In the present invention, unless otherwise specified, the stainless steel in the embodiments of the present invention is 304 stainless steel.

[0044] In the present invention, unless otherwise specified, the material of the sample rack in the embodiments of the present invention is polytetrafluoroethylene.

[0045] In the present invention, unless otherwise specified, the tubular reactor in the embodiments of the present invention is a borosilicate glass tubular reactor.

[0046] In the present invention, unless otherwise specified, the outer shell material of the one-way valve in the embodiments of the present invention is polypropylene, and the gasket material is silicone rubber.

[0047] In the present invention, unless otherwise specified, the material of the hose in the embodiments of the present invention is silicone, and the material of the thermal insulation layer is sponge.

[0048] Example 1

[0049] Referring to Figure 1 - Figure 2 As shown, the culture system for thermophilic bacteria biofilm in flowing dairy matrix of this embodiment includes:

[0050] Milk supply tank 2: for storing and supplying dairy products;

[0051] Power device 3: connected to the milk supply tank 2, and the power device 3 is a peristaltic pump for transporting the dairy products in the milk supply tank 2 to the biofilm culture unit;

[0052] Biofilm culture unit: includes a tubular reactor 5 and an inner ring stainless steel layer 8 arranged inside the tubular reactor 5; a sample rack 7 for placing stainless steel samples 6 is provided on the inner surface of the inner ring stainless steel layer 8; the input end of the biofilm culture unit is connected to the power device 3, and the output end is connected to the one-way valve 13; the inner diameter of the tubular reactor 5 is 4 cm and the length is 25 cm; both ends of the tubular reactor 5 are provided with a first disassembly port 11 and a second disassembly port 12 for placing and sampling samples; the inner surface area of the inner ring stainless steel layer 8 is equivalent to that of the inner surface of the tubular reactor 5; the shape of the stainless steel sample 6 is circular or square, the diameter of the circular stainless steel sample 6 is 1.5 cm, and the side length of the square stainless steel sample 6 is 1.5 cm; the sample rack 7 is provided with independently detachable or row-connected non-detachable grooves, and the shape of the grooves matches the shape of the stainless steel sample 6. The biofilm culture unit is in an inclined state, and the inclination angle is 25°, which is realized by a low support 9 and a high support 10.

[0053] One-way valve 13: for controlling the single flow direction of dairy products and preventing the reverse flow and pollution of dairy products to the biofilm culture unit;

[0054] Sampling port 14: connected to the one-way valve 13, and the sampling port 14 is a three-way switch valve for controlling the flow direction of dairy products to the sampling bottle or the waste liquid recovery tank 15.

[0055] There is also a water bath heating device 1 and a heating jacket heating device; the water bath heating device 1 is used to heat the milk supply tank 2; the heating jacket heating device includes a flexible heating jacket 4 and a temperature control device, and the flexible heating jacket 4 is wrapped on the surface of the tubular reactor 5; the heating jacket heating device is used to heat the tubular reactor 5.

[0056] The specific connection method of each unit is connected by a hose 16, and a heat insulation layer 17 with a thickness of 0.5 cm is coated on the surface of the hose 16.

[0057] Example 2

[0058] Refer to Figure 3 As shown, the method for culturing thermophilic bacteria biofilm in the flowing dairy matrix of this example uses the culturing system of Example 1 as the generating device, and specifically includes the following steps:

[0059] S1. Strain activation: Bacillus licheniformis and Clostridium thermoanaerobium (inoculation ratio 1:1) are inoculated into milk dairy products at a volume ratio of 1%, and cultured with shaking at 55 °C for 8 h to obtain dairy products containing thermophilic bacteria (the thermophilic bacteria are in a floating state, that is, the floating bacteria when the biofilm is not formed and can swim freely).

[0060] S2. Device assembly: First, assemble the culturing system according to Example 1. After the assembly is completed, prepare several stainless steel sample pieces and sterilize them by moist heat at 121 °C for 15 min; then take out the sample rack 7 and use sterile forceps to place the stainless steel sample pieces on the sample rack 7 in sequence, one piece in each groove; finally, put the sample rack 7 back into the tubular reactor 5, reconnect the two ends, and wrap it in the flexible heating jacket 4.

[0061] S3. Dynamic culture: Load the dairy products containing thermophilic bacteria into the milk supply tank 2, set the heating temperatures of the water bath heating device 1 and the flexible heating jacket 4 heating device to 55 °C, and the flow rate of the power device 3 to 5 mL / min. The dairy products containing thermophilic bacteria are transported to the tubular reactor 5 in the biofilm culturing unit by the power device 3 for dynamic culture for 12 h;

[0062] S4. Obtain sample pieces: After continuous culture for 12 h, use sterile forceps to take out the stainless steel sample pieces on the sample rack 7, and the stainless steel sample pieces adhered with thermophilic bacteria can be obtained.

[0063] S5. Maturation and detachment: First, re-prepare the culture system and stainless steel specimens with reference to S2. Place several stainless steel specimens with thermophilic bacteria adhered obtained in S4 and several stainless steel specimens on the specimen rack 7. Then, load the sterilized milk dairy product into the milk supply tank 2, set the heating temperature of the water bath heating device 1 and the flexible heating sleeve 4 heating device to 55°C, and the flow rate of the power device 3 to 5 mL / min. Transport the sterilized milk dairy product to the tubular reactor 5 of the biofilm culture unit through the power device 3 for cultivation. After continuous cultivation for 12 h, use sterile forceps to take out the stainless steel specimens with thermophilic bacteria adhered from S4 on the specimen rack 7, and then the detachment situation of the thermophilic bacteria biofilm 18 on the stainless steel specimens with thermophilic bacteria adhered can be measured.

[0064] S6. Secondary adhesion: Or after continuous cultivation for 12 h, use sterile forceps to take out the stainless steel specimens from S5 on the specimen rack 7, that is, the stainless steel specimens with secondary adhesion of thermophilic bacteria, and then the degree of influence of the stainless steel specimens by the stainless steel specimens with thermophilic bacteria adhered, that is, the secondary adhesion situation of the thermophilic bacteria biofilm 18, can be measured.

[0065] Example 3: Using Thermophilic Bacteria Biofilm for Total Colony Count

[0066] (1) Characterization of the ability of thermophilic bacteria to form biofilm: Based on Example 2, during the assembly of the device in S2, place 10 stainless steel specimens on the specimen rack in sequence. Then, during the 12 h of dynamic cultivation, take out 2 - 3 stainless steel specimens every 4 h. Place the stainless steel specimens in a 50 mL centrifuge tube containing 5 mL of sterile normal saline and 5 g of sterile glass beads with a diameter of 3 mm using sterile forceps, tighten the lid, and vortex for 1 min. Pipette 50 μL of the bacterial-containing liquid and spread it on the tryptic soy agar medium, and incubate overnight at 55°C, then count the logarithm of the total colony number (log CFU). With time (h) as the abscissa and the logarithm of the colony-forming unit per unit area (calculate the area of each stainless steel specimen according to the diameter or side length of the stainless steel specimen, in cm 2 counted) (logCFU / cm 2 ) as the ordinate to plot a graph to characterize the ability of thermophilic bacteria to form biofilm on the stainless steel surface in the flowing dairy product matrix. The results are as shown in Figure 4 (A). It can be seen from Figure 4 (A) that in the milk matrix, the starting formation time of the above-mentioned dual-bacteria biofilm adhered to the stainless steel surface developed from the planktonic bacterial liquid (in the state of not forming biofilm) is between 4 h and 8 h after inoculation, and shows a trend that the adhesion amount gradually increases with the prolongation of the cultivation time. At the 8th h, the total number of thermophilic bacteria per unit area is about on the order of 10 3 CFU, while at the 12th h, the total number of thermophilic bacteria per unit area increases to 10 4The order of magnitude of CFU. It shows that by using the cultivation method of the present invention, biofilm specimens of thermophilic bacteria adhering to the surface of stainless steel material in a flowing and renewed dairy matrix can be obtained during continuous cultivation, and their dynamic formation ability can be effectively evaluated.

[0067] (2) The shedding situation of mature thermophilic bacteria biofilm: Based on Example 2, during the mature shedding process in S5, 10 stainless steel specimens adhered with thermophilic bacteria were successively placed on the specimen rack. Then, during the 12 hours of mature shedding, 2 - 3 stainless steel specimens adhered with thermophilic bacteria were taken out every 4 hours. The stainless steel specimens adhered with thermophilic bacteria were placed in a 50 mL centrifuge tube containing 5 mL of sterile normal saline and 5 g of sterile glass beads with a diameter of 3 mm using sterile forceps, the lid was tightened, and vortexed for 1 minute. 50 μL of the bacterial-containing liquid was aspirated and spread on tryptic soy agar medium, and after overnight cultivation at 55 °C, the logarithm of the total number of colonies (logCFU) was counted. Using time (h) as the abscissa and the logarithm of the colony forming unit per unit area (calculate the area of each stainless steel specimen according to the diameter or side length of the stainless steel specimen, in cm 2 counted) (log CFU / cm 2 ) as the ordinate to plot a graph to characterize the shedding situation of mature thermophilic bacteria biofilm, and the results are as shown in Figure 4 (B). It can be seen from Figure 4 (B) that when the mixed adhesion dual-bacteria sample from mature biofilm is cultured in milk medium for the second time, it will show a dynamic development and balance process of shedding first and then continuing to expand. Within 4 hours of cultivation, the adhered bacteria on the mature thermophilic bacteria biofilm experienced the first shedding process, and the total number of bacteria decreased by nearly 1 order of magnitude (log CFU / cm 2 ); within 4 h - 8 h, the adhered bacteria experienced the second round of reproduction, and the total number of bacteria increased by nearly 3 orders of magnitude, reaching 10 5 log CFU / cm 2 , significantly exceeding the total number at the 12th hour in (1), showing stronger growth and adhesion ability; at the 12th hour, the total number of adhered bacteria decreased slightly again, indicating that the second shedding cycle may start. It shows that the thermophilic bacteria from biofilm have the physiological characteristics of periodic maturation, shedding, and adhesion, and their adhesion ability is stronger than that of planktonic bacteria. The cultivation system and cultivation method of the present invention provide a way to study these physiological characteristics of thermophilic bacteria, and the obtained data point out an important direction for the prevention and control of thermophilic bacteria biofilm in the dairy processing process.

[0068] (3) Secondary adhesion ability of thermophilic bacteria biofilm: Based on Example 2, during the process of maturation and detachment in S5, one stainless steel sample piece adhered with thermophilic bacteria and nine stainless steel sample pieces were successively placed on the sample rack. Then, during the 12 hours of secondary adhesion, every 4 hours, 2 - 3 stainless steel sample pieces were taken out. The stainless steel sample pieces were placed into a 50 mL centrifuge tube containing 5 mL of sterile physiological saline and 5 g of sterile glass beads with a diameter of 3 mm using sterile forceps. The lid was tightened and vortexed for 1 minute. 50 μL of the bacteria-containing liquid was aspirated and spread on the tryptic soy agar medium, and after overnight culture at 55 °C, the logarithm of the total number of colonies (log CFU) was counted. With time (h) as the abscissa and the logarithm of the number of colonies per unit area (calculate the area of each stainless steel sample piece according to the diameter or side length of the stainless steel sample piece, in cm 2 counted) per unit (log CFU / cm 2 ) as the ordinate, a graph was plotted to characterize the secondary adhesion ability of the thermophilic bacteria biofilm. The results are shown in Figure 4 (C). As can be seen from Figure 4 (C), when the mixed adhesion dual-bacteria sample from the mature thermophilic bacteria biofilm was cultured in the milk medium for the second round, it was able to adhere to the clean stainless steel sample pieces. This ability is the secondary adhesion ability of the thermophilic bacteria biofilm. Its overall trend is similar to the ability of planktonic bacteria to form biofilms ( Figure 4 (A)), showing an increasing trend within 4 h - 12 h. However, the adhesion ability of the bacteria from the biofilm is stronger. At the 12th hour, it exceeds 10 5 log CFU / cm 2 , much higher than the adhesion ability of planktonic bacteria at the 12th hour (about 10 4 log CFU / cm 2 ). It shows that the detached biofilm bacteria have the ability to find new habitats, and this secondary adhesion ability is the key reason for the cross-contamination of thermophilic bacteria in dairy processing. The method of the present invention provides a new strategy and technology for studying the secondary adhesion ability of biofilms.

[0069] Example 4 Observation of Thermophilic Bacteria Biofilm by Laser Confocal Microscopy Using Specific Fluorescent Probe Labeling

[0070] Specific fluorescence probe labeling requires designing specific fluorescence probes according to the 16S DNA sequence of the target strain. Taking the observation of the lichen bacillus sample as an example, the specific probe is linked to the CY3 fluorescent dye. Based on the mixed biofilm sample of lichen bacillus and thermophilic yellow anaerobic bacillus adhered to the surface of the stainless steel sample prepared in Example 3(3), only the lichen bacillus among them is subjected to specific probe hybridization. The hybridization method is as follows: rinse the sample with 0.85% normal saline to remove planktonic bacteria, and leave it to dry naturally; fix the biofilm sample with 96% ice ethanol at 4°C overnight; rinse the sample, use 1mg / mL lysozyme to lyse the biofilm cells at room temperature for 10 minutes, and rinse again; dehydrate the sample with 50%, 80%, and 96% ethanol gradiently, 3 minutes each time, and leave it to dry for later use; prepare the hybridization solution: 0.9mol / L NaCl, 20mmol / L Tris-HCl, 0.1% SDS, 30% formamide, 5ng / μL probe; immerse the biofilm sample in the hybridization solution and hybridize at 46°C for 3 hours, then rinse; prepare the elution solution: 20mmol / L Tris-HCl, 5mmol / L EDTA, 102mmol / L NaCl, 0.01% SDS; elute the sample at 48°C for 15 minutes, rinse, dry, and mount the sample; observe the morphology of the lichen bacillus biofilm sample under a laser confocal microscope at the excitation and emission wavelengths of 514nm / 566nm. The results are as Figure 5 shown. It can be seen from Figure 5 that the main morphology of lichen bacillus on the stainless steel surface shows a monolayer distribution, with a relatively large population density, but the cells are relatively dispersed and do not actively aggregate. Using the lichen bacillus in the mixed biofilm sample prepared by the present invention for laser confocal microscopy observation with specific fluorescence probe labeling can clearly reflect important information such as the morphology of the adhered strain, population density, and distribution of this strain on the stainless steel surface, providing an important research method for qualitative and semi-quantitative characterization of a single strain in the mixed biofilm.

[0071] Obviously, the above embodiments are only examples given for clear illustration and are not limitations on the implementation manners. For those of ordinary skill in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation manners here. And the obvious changes or modifications derived therefrom are still within the protection scope of the present invention.

Claims

1. A system for culturing thermophilic biofilm in a flowing dairy matrix, characterized in that: include: Milk supply tank: used for storing and supplying milk products; a power device connected to the milk supply tank, used for transporting the milk products in the milk supply tank to the biofilm culture unit; The biofilm culture unit comprises a tubular reactor and a ring inner wall stainless steel layer arranged in the tubular reactor; the inner surface of the ring inner wall stainless steel layer is provided with a sample rack for placing samples; the input end of the biofilm culture unit is connected to the power device, and the output end is connected to the one-way valve; One-way valve: used to control the single flow direction of dairy products; the sampling port, connected to the one-way valve, is used to control the flow of dairy products to the sampling bottle or the waste liquid recovery tank.

2. The cultivation system for thermophilic biofilm in a flowing dairy matrix according to claim 1, characterized in that: It also includes a water bath heating device and a heating jacket heating device; the water bath heating device is used to heat the milk supply tank; the heating jacket heating device is used to heat the tubular reactor.

3. The cultivation system for thermophilic biofilm in a flowing dairy matrix according to claim 1, characterized in that: The inner diameter of the tubular reactor is 2 cm-6 cm, and the length is 15 cm-30 cm; The shape of the sample is round or square, the diameter of the round sample is 1cm-2cm, and the side length of the square sample is 1cm-2cm.

4. The cultivation system for thermophilic biofilm in a flowing dairy matrix according to claim 1, characterized in that: The biofilm culture unit is in an inclined state, and the inclination angle is 10°-45°.

5. A method for culturing thermophilic biofilm in a flowing dairy matrix, characterized in that: The culture system according to any one of claims 1 to 4 is used as a generating device, comprising the following steps: S1. Place the first sample on the sample rack and install it into the biofilm culture unit; S2, loading the milk product containing thermophilic bacteria into the milk supply tank, and setting the heating temperature of the water bath heating device and the heating jacket heating device; S3, transporting the thermophilic bacteria-containing dairy product to the biofilm culture unit for culture through a power device, and taking out the first sample; S4, re-prepare the culture system, place the second sample and the first sample on the sample rack, and install them into the biofilm culture unit; S5, loading the milk product without thermophilic bacteria into the milk supply tank, and setting the heating temperature of the water bath heating device and the heating jacket heating device; S6. The milk product without thermophilic bacteria is transported to the biofilm culture unit for culture by a power device, and the first sample is taken out to determine the biofilm shedding situation; and / or, the second sample is taken out to determine the degree to which the second sample is affected by the first sample.

6. The method for culturing thermophilic biofilm in a flowing dairy matrix according to claim 5, characterized in that: In S2, the thermophilic bacteria are selected from one or more of Bacillus licheniformis, Bacillus thermophilus, Bacillus stearothermophilus and Bacillus subtilis.

7. The method for culturing thermophilic biofilm in a flowing dairy matrix according to claim 5, characterized in that: In S2, the thermophilic bacteria-containing dairy product is further subjected to a bacteria activation treatment before use; the bacteria activation is carried out by shaking culture at 54° C.-56° C. for 6 h-10 h.

8. The method for culturing thermophilic biofilm in a flowing dairy matrix according to claim 5, characterized in that: The heating temperatures of the water bath heating device and the heating jacket heating device are independently 50°C to 70°C.

9. The method for culturing thermophilic biofilm in a flowing dairy matrix according to claim 5, characterized in that: The delivery rate of the power device is independently 1 mL / min-100 mL / min.

10. The method for culturing thermophilic biofilm in a flowing dairy matrix according to claim 5, characterized in that: The culture time is independently 6h-24h.

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